1. Field of the Invention
The present invention relates to the field of cardiovascular and other diseases. More particularly, the present invention concerns compositions and methods of identification and use of isoform-selective activators or inhibitors of cyclic nucleotide phosphodiesterase PDE3 (‘PDE3’). Other embodiments of the invention concern high-throughput screening for novel pharmaceuticals directed against PDE3 isoforms. In certain embodiments, the compositions and methods disclosed herein are of use for treatment of cardiomyopathy, pulmonary hypertension and related conditions.
2. Description of Related Art
PDE3 cyclic nucleotide phosphodiesterases hydrolyze cAMP and cGMP and thereby modulate cAMP- and cGMP-mediated signal transduction (Shakur et al., 2000a). These enzymes have a major role in the regulation of contraction and relaxation in cardiac and vascular myocytes. PDE3 inhibitors, which raise intracellular cAMP and cGMP content, have inotropic effects attributable to the activation of cAMP-dependent protein kinase (‘PKA’) in cardiac myocytes and vasodilatory effects attributable to the activation of cGMP-dependent protein kinase (‘PKG’) in vascular myocytes (Shakur et al., 2000a). When used in the treatment of dilated cardiomyopathy, PDE3 inhibitors such as milrinone, enoximone and amrinone initially elicit favorable hemodynamic responses, but long-term administration increases mortality by up to 40% (Nony et al., 1994). This linkage of short-term benefits of PDE3 inhibition to deleterious effects on long-term survival in dilated cardiomyopathy is problematic in the field of cardiovascular therapeutics.
Clinical trials of the use of β-adrenergic receptor agonists, which, like PDE3 inhibitors, increase intracellular cAMP content in cardiac myocytes, were terminated prior to completion because of increased mortality in treated patients, while β-adrenergic receptor antagonists, which reduce intracellular cAMP content, have been shown to improve long-term survival despite initially adverse hemodynamic effects. These findings suggest that both the short-term benefits and long-term adverse effects of PDE3 inhibition are attributable to increases in intracellular cAMP content in cardiac myocytes (Movsesian, 1999).
Upon activation by cAMP, PKA phosphorylates a large number of proteins in separate intracellular compartments that are involved in a number of processes, including but not limited to contraction and relaxation, glycogen metabolism, gene transcription, intracellular Ca2+ cycling and signal autoregulation. Phosphorylation of cAMP-response element-binding protein (CREB), for example, activates the transcription of genes containing cAMP response elements (Shaywitz and Greenberg, 1999).
Another example of cAMP effects is the phosphorylation of phospholamban, which relieves its inhibition of SERCA2, the Ca2+-transporting ATPase of the sarcoplasmic reticulum (Simmerman and Jones, 1998). Ablation of phospholamban in muscle LIM protein (MLP)−/− mice with dilated cardiomyopathy results in the restoration of normal chamber size and contractility (Minamisawa et al., 1999), suggesting that phospholamban phosphorylation may also be beneficial in cardiomyopathy.
Other substrates phosphorylated by PKA may contribute to adverse effects on long-term survival. Phosphorylation of L-type Ca2+ channels increases their open probability and may be arrhythmogenic (Fischmeister and Hartzell, 1990), while phosphorylation of proteins in the mitogen-activated protein kinase (MAP kinase) cascade may alter myocardial gene transcription so as to speed the progression of the disease (Cook and McCormick, 1993; Lazou et al., 1994).
Raising cAMP content in cardiac myocytes via mechanisms such as activation of β1-adrenergic, β2-adrenergic or prostaglandin receptors or non-selective phosphodiesterase inhibition by isobutylmethylxanthine affects cAMP content differentially in intracellular compartments represented in cytosolic and microsomal fractions of cardiac muscle, resulting in different patterns of protein phosphorylation and different physiologic responses (Hayes et al., 1980; Xiao and Lakatta, 1993; Xiao et al., 1994; Rapundalo et al., 1989; Jurevicius and Fischmeister, 1996). These considerations are particularly relevant to the pathophysiology of dilated cardiomyopathy, in which receptor-mediated and receptor-independent reductions in cAMP generation are prominent features (Movsesian, 1999; Lutz, et al., 2001). Comparison of cytosolic cAMP content in cytosolic and microsomal fractions between failing and non-failing hearts shows greater reduction in cAMP content in microsomal fractions of failing myocardium than in cytosolic fractions (Bohm, 1994).
The phosphorylation of individual substrates of PKA is differentially regulated in response to extracellular signals. Evidence for differential regulation comes from experiments examining the effects of stimulating adenylate cyclase activity and cAMP formation via β1-adrenergic, β2-adrenergic or PGE1 receptors. Activation of β-adrenergic receptors increases cAMP content in both cytosolic and microsomal fractions of cardiac myocytes and elicits contractile responses, while activation of PGE1 receptors increases cytosolic but not microsomal cAMP content and evokes no contractile response (Hayes et al., 1980; Buxton and Brunton, 1983). Increases in the amplitude of intracellular Ca2+ transients in response to β1-adrenergic receptor activation correlate with changes in microsomal cAMP content and are accompanied by increases in phospholamban phosphorylation. Conversely, activation of β2-adrenergic receptors results in an increase in the amplitude of intracellular Ca2+ transients that does not correlate with changes in microsomal cAMP content and occurs without increases in phospholamban phosphorylation (Hohl and Li, 1991; Xiao et al., 1993, 1994). Thus, activation of different receptors linked to cAMP metabolism can elicit different responses in cardiac tissues.
β-adrenergic receptor stimulation and nonselective phosphodiesterase inhibition have different effects on cAMP-activated protein phosphorylation in cardiac myocytes (Rapundalo et al., 1989; Jurvicius and Fischmeister, 1996) that are relevant to the pathophysiology of dilated cardiomyopathy. In that condition, a down-regulation of β1-adrenergic receptors and an uncoupling of β-adrenergic receptor occupancy and adenylate cyclase stimulation (attributable to increases in β-adrenergic receptor kinase, Gai and nucleoside diphosphate kinase) contribute to an impairment in cAMP generation (Movsesian, 1999; Lutz et al., 2001). Studies of cAMP content in cytosolic and microsomal fractions of failing and non-failing hearts demonstrate a far greater reduction in cAMP content in microsomal fractions than in cytosolic fractions of failing myocardium (Bohm et al., 1994). Taken together, these results indicate that cAMP content in different intracellular compartments can be selectively regulated to invoke different responses reflecting the phosphorylation of different substrates of PKA. Further, this regulation is altered in dilated cardiomyopathy.
Different isoforms of PDE3 are expressed in cardiac and vascular myocytes and are localized to different intracellular compartments. The different PDE3 isoforms may differ in their regulation by PKA and PKB (protein kinase B, also known as Akt). PKB, a downstream effector of insulin-like growth factors, is an anti-apoptotic mediator in cardiac myocytes (Fujio et al., 2000; Matsui et al., 1999; Wu et al., 2000). PKB may also be involved in proliferative responses in vascular myocytes (Rocic and Lucchesi, 2001; Duan et al., 2000; Sandirasegarane et al., 2000). These findings suggest that different PDE3 isoforms may be involved in cell- and compartment-selective responses to different signals that have been implicated in the pathophysiology of dilated cardiomyopathy and/or pulmonary hypertension. Different PDE3 isoforms in cardiac and vascular myocytes may regulate functionally distinct pools of cAMP and cGMP involved in the phosphorylation of different substrates of PKA and PKG, and these isoforms may be regulated in response to different extracellular signals.